Sequence and codon recognition of bean mitochondria and chloroplast tRNAsTrp: evidence for a high degree of homology.

Bean mitochondria and chloroplast tRNAsTrp, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, have been sequenced using post-labeling techniques. The high degree of sequence homology between bean mitochondria and chloroplast tRNAsTrp shows that these two tRNAs are coded for by closely related genes which have probably evolved from a common ancestor gene. The anticodon of bean mitochondria tRNATrp is CmCA, which can recognize UGG (the codon for tryptophan in the universal code) and is complementary neither to UGA (which codes for tryptophan in mammalian and yeast mitochondria) nor to CGG (which could be a tryptophan codeword in plant mitochondria).     

Structure and function of tryptophan tRNA from wheat germ.

The coding properties of tRNATrp from yeast and wheat germ were studied. Unlike E. coli tRNATrp or mitochondrial tRNATrp, eukaryotic tRNATrp did not recognize the UGA codon in vitro. The sequence of wheat germ tRNATrp as determined by [32P] post-labelling techniques is: [sequence in text] The interesting features are: (i) Presence of a C11:G24 base pair in contrast to the U11:G24 in E. coli Su- tRNATrp. (ii) The anticodon sequence is -CmCA- compared to -CCA- in E. coli tRNATrp. (iii) Lack of a hypermodified base i6A adjacent to the 3'-end of the anticodon. (iv) Presence of -T psi CG- sequence instead of -psi psi CG- sequence present in mammalian tRNATrp.     

Locations and sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC): the tRNAGly contains only two base-pairs in the D stem.

The nucleotide sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC) have been determined. None of these genes contains an intron. One unusual feature is that the tRNAGly contains only two base-pairs (A-U, G-U) in the D stem. These four tRNA genes were located in the known physical map of tobacco chloroplast DNA. Hybridization analysis to chloroplast tRNA revealed that all four tRNA genes are transcribed in vivo.     

The chloroplast infA gene with a functional UUG initiation codon

All chloroplast genes reported so far possess ATG start codons and sometimes GTGs as an exception. Sequence alignments suggested that the chloroplast infA gene encoding initiation factor 1 in the green alga Chlorella vulgaris has TTG as a putative initiation codon. This gene was shown to be transcribed by RT-PCR analysis. The infA mRNA was translated accurately from the UUG codon in a tobacco chloroplast in vitro translation system. Mutation of the UUG codon to AUG increased translation efficiency approximately 300-fold. These results indicate that the UUG is functional for accurate translation initiation of Chlorella infA mRNA but it is an inefficient initiation codon.     

Selection on the codon bias ofChlamydomonas reinhardtii chloroplast genes and the plantpsbA gene

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translatedpsbA gene which codes for the photosystem II D1 protein. The codon usage of plantpsbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green algaChlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plantpsbA andChlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plantpsbA andChlamydomonas chloroplast genes may be the result of differences in second position composition.     

The effect of point mutations affecting Escherichia coli tryptophan tRNA on anticodon-anticodon interactions and on UGA suppression

tRNA species in Escherichia coli that translate codons starting with U contain 2-methyl-thio-N6-isopentenyl-adenosine in position 37, 3' adjacent to the anticodon. The role of this hypermodification in protein synthesis and trp operon attenuation has been investigated. Temperature-jump relaxation methods have been applied to study the interaction between E. coli tRNAPro, with anticodon VGG (V is uridine-5-oxyacetic acid) complementary to that of tRNATrp, and three species of E. coli tRNATrp: wild type tRNATrp (with ms2i6A37 and G24), UGA suppressor tRNATrp (with ms2i6A37 and A24 in the dihydrouridine stem but the same anticodon CCA), and the same suppressor molecule but ms2i6A-deficient as a result of the mutation miaA. Complex formation between tRNAPro and ms2i6A-containing tRNATrp shows thermodynamic parameters close to those found for several other pairs of tRNA with complementary anticodons. However, ms2i6A-deficient tRNATrp makes less stable complexes with tRNAPro, which dissociate eightfold faster. No effect on the complementary anticodon interaction of the mutation in the dihydrouridine stem can be detected. When the tRNA analogous to the opal codon, E. coli tRNASerIV (anticodon VGA) replaces tRNAPro in similar experiments, very weak complexes are observed with both normally hypermodified species of tRNATrp, the wild type and UGA suppressor; these show a lifetime about 50-fold shorter than with tRNAPro, but are again similar. No complex formation is detectable with the ms2i6A-deficient species. This may explain why the hypermodification is necessary for the efficient suppression of the UGA terminator of Q beta coat protein in vitro. The data on complexes with tRNAPro suggest that deficiency in ms2i6A may also reduce the efficiency of UGG reading. Thus, miaA may affect trp operon attenuation by slowing translation of the tandem UGG codons in the leader sequence. Temperature-jump differential spectra suggest that ms2i6 stabilizes the anticodon interaction by improved stacking of base 37.     

Synonymous and Nonsynonymous Substitution Rates in Diatoms: A Comparison Between Chloroplast and Nuclear Genes

Rates of synonymous and nonsynonymous nucleotide substitutions and codon usage bias (ENC) were estimated for a number of nuclear and chloroplast genes in a sample of centric and pennate diatoms. The results suggest that DNA evolution has taken place, on an average, at a slower rate in the chloroplast genes than in the nuclear genes: a rate variation pattern similar to that observed in land plants. Synonymous substitution rates in the chloroplast genes show a negative association with the degree of codon usage bias, suggesting that genes with a higher degree of codon usage bias have evolved at a slower rate. While this relationship has been shown in both prokaryotes and multicellular eukaryotes, it has not been demonstrated before in diatoms. Received: 3 June 1998 / Accepted: 11 August 1998     

Chloroplast DNA codon use: Evidence for selection at the psb A locus based on tRNA availability

Codon use in the three sequenced chloroplast genomes (Marchantia, Oryza, and Nicotiana) is examined. The chloroplast has a bias in that codons NNA and NNT are favored over synonymous NNC and NNG codons. This appears to be a consequence of an overall high A + T content of the genome. This pattern of codon use is not followed by the psb A gene of all three genomes and other psb A sequences examined. In this gene, the codon use favors NNC over NNT for twofold degenerate amino acids. In each case the only tRNA coded by the genome is complementary to the NNC codon. This codon use is similar to the codon use by chloroplast genes examined from Chlamydomonas reinhardtii. Since psb A is the major translation product of the chloroplast, this suggests that selection is acting on the codon use of this gene to adapt codons to tRNA availability, as previously suggested for unicellular organisms.     

The forms of tRNATrp found in avian sarcoma virus and uninfected chicken cells have structural identity but functional distinctions

We have determined the complete nucleotide sequence of the avian tRNATrp which serves as primer for avian retrovirus DNA synthesis by the viral polymerase. The sequence is identical to that reported for tRNATrp present in uninfected avian cells (Harada, F., Sawyer, R. C., and Dahlberg, J. E. (1975) J. Biol. Chem. 250, 3487-3497). Although there appears to be only a single species of tRNATrp in avian cells, two functionally different forms within the population can be distinguished. We show that the tRNATrp isolated from virions can act in vitro as an efficient suppressor for UGA. The suppressor activity is roughly 3-fold greater with viral tRNATrp than with cellular tRNATrp. In addition, it has been reported (Panet, A., Haseltine, W. A., Baltimore, D., Peters, G., Harada, F., and Dahlberg, J. E. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2535-2539) that the viral polymerase can bind 100% of viral tRNATrp, but only 30% of cellular tRNATrp. Hence, avian retroviruses seem to selectively incorporate and utilize only one of these forms. Since the nucleotide sequence and nucleoside modifications are identical between viral and cellular tRNATrp, two conformations of avian tRNATrp may exist which can influence several biological activities of the molecule.     

Mutual conformational changes of tryptophanyl-tRNA synthetase and tRNATrp in the course of their specific interaction

tRNATrp (beef, yeast) is capable of accelerating limited tryptic hydrolysis of the N-terminal part in the polypeptide chains of dimeric beef pancreas tryptophanyl-tRNA synthetase; it can also eliminate the protective effect of tryptophanyl adenylate on the enzyme proteolysis. The effect of tRNA on the proteolysis is manifested even when the 3'-CCA terminus is removed. It has been concluded that the conformation of the synthetase changes when it forms a complex with tRNATrp. Yeast tRNATrp lacking the 3'-half of the acceptor stem can still interact with the synthetase and, to certain extent, induces changes in the conformation of the latter. The susceptibility of single-stranded and double-stranded regions of tRNATrp to cleavage with endonucleases has been studied, and the results are indicative of the fact that, regardless of considerable differences in the nucleotide sequence of yeast and beef tRNATrp, their three-dimensional structures are similar. This fact is consistent with the finding that parameters for the interaction of these tRNAsTrp with beef tryptophanyl-tRNA synthetase are rather close. The three-dimensional structure of tRNATrp is altered when the enzyme forms a complex with it, as seen from (a) a change in the circular dichroic spectrum and (b) an elevated susceptibility of the anticodon and, apparently, acceptor stems to cleavage with nuclease. The conversion of exposed cytidine residues in tRNATrp into uridine residues results in a loss of the acceptor activity; the capability to accelerate limited tryptic hydrolysis of tryptophanyl-tRNA synthetase is also lost although the enzyme-substrate complex, as seen from circular dichroic spectra, can still be formed. The conversion of cytosine in the anticodon stem into uracil modifies the conformation of the anticodon stem. The anticodon arm (including the anticodon) and the acceptor stem play an essential role in the interaction between tRNATrp and tryptophanyl-tRNA synthetase.     

A role for initiation codon context in chloroplast translation

To study the role of initiation codon context in chloroplast protein synthesis, we mutated the three nucleotides immediately upstream of the initiation codon (the -1 triplet) of two chloroplast genes in the alga Chlamydomonas reinhardtii. In prokaryotes, the -1 triplet has been proposed to base pair with either the 530 loop of 16S rRNA or the extended anticodon of fMet-tRNA. We found that in vivo, none of the chloroplast mutations affected mRNA stability. However, certain mutations did cause a temperature-sensitive decrease in translation and a more dramatic decrease at room temperature when combined with an AUU initiation codon. These mutations disrupt the proposed extended base pairing interaction with the fMet-tRNA anticodon loop, suggesting that this interaction may be important in vivo. Mutations that would still permit base pairing with the 530 loop of the 16S rRNA also had a negative effect on translation, suggesting that this interaction does not occur in vivo. Extended base pairing surrounding the initiation codon may be part of a mechanism to compensate for the lack of a classic Shine-Dalgarno rRNA interaction in the translation of some chloroplast mRNAs.     

Codon use and the rate of divergence of land plant chloroplast genes

Codon fitnesses for chloroplast genes were estimated using the relative synonymous codon use of psbA, which has a different pattern of codon use than other chloroplast genes and is the major translation product of the chloroplast. These estimates were used to calculate the codon adaptation index (CAI) of chloroplast genes from Marchantia polymorpha, Nicotiana tabacum, and Chlamydomonas reinhardtii. The genes with the highest CAI values in M. polymorpha correspond to those that are expressed at the highest levels. The rate of divergence between M. polymorpha and both C. reinhardtii and N. tabacum is inversely related to the CAI value of the M. polymorpha gene. The data suggest that selection is acting on the synonymous codon use of the highly expressed genes of the M. polymorpha chloroplast genome. The data set is inconclusive about N. tabacum genes, but, as there is a weaker correspondence between CAI value and expression level, it suggests that selection is not operating in this lineage.      

Comparative studies on codon usage pattern of chloroplasts and their host nuclear genes in four plant species

A detailed comparison was made of codon usage of chloroplast genes with their host (nuclear) genes in the four angiosperm speciesOryza sativa, Zea mays, Triticum aestivum andArabidopsis thaliana. The average GC content of the entire genes, and at the three codon positions individually, was higher in nuclear than in chloroplast genes, suggesting different genomic organization and mutation pressures in nuclear and chloroplast genes. The results of Nc-plots and neutrality plots suggested that nucleotide compositional constraint had a large contribution to codon usage bias of nuclear genes inO. sativa, Z. mays, andT. aestivum, whereas natural selection was likely to be playing a large role in codon usage bias in chloroplast genomes. Correspondence analysis and chi-test showed that regardless of the genomic environment (species) of the host, the codon usage pattern of chloroplast genes differed from nuclear genes of their host species by their AU-richness. All the chloroplast genomes have predominantly A- and/or U-ending codons, whereas nuclear genomes have G-, C- or U-ending codons as their optimal codons. These findings suggest that the chloroplast genome might display particular characteristics of codon usage that are different from its host nuclear genome. However, one feature common to both chloroplast and nuclear genomes in this study was that pyrimidines were found more frequently than purines at the synonymous codon position of optimal codons.     

Comparative Analysis of Codon Usage Patterns Among Mitochondrion, Chloroplast and Nuclear Genes in Triticum aestivum L.

In many organisms, the difference in codon usage patterns among genes reflects variation in local base compositional biases and the intensity of natural selection. In this study, a comparative analysis was performed to investigate the characteristics of codon bias and factors in shaping the codon usage patterns among mitochondrion, chloroplast and nuclear genes in common wheat （Triticum aestivum L.）. GC contents in nuclear genes were higher than that in mitochondrion and chloroplast genes. The neutrality and correspondence analyses indicated that the codon usage in nuclear genes would be a result of relative strong mutational bias, while the codon usage patterns of mitochondrion and chloroplast genes were more conserved in GC content and influenced by translation level. The Parity Rule 2 （PR2） plot analysis showed that pyrimidines were used more frequently than purines at the third codon position in the three genomes. In addition, using a new alterative strategy, 11, 12, and 24 triplets were defined as preferred codons in the mitochondrion, chloroplast and nuclear genes, respectively. These findings suggested that the mitochondrion, chloroplast and nuclear genes shared particularly different features of codon usage and evolutionary constraints.     

毛茛科和芍药科叶绿体基因组密码子特征和系统发育比较

为研究和比较毛茛科和芍药科叶绿体基因组密码子使用模式和系统进化关系,以完成测序的毛茛科33种植物、芍药科7种植物叶绿体基因组为材料,采用分析软件CodonW在线软件CUSP和R软件对叶绿体基因进行密码子特征分析。用MAFFT软件,MEGA软件进行系统发育分析。研究结果表明芍药科植物叶绿体基因组和毛茛科植物（耧斗菜属除外）叶绿体基因组高频密码子一致性高,具有29个高频密码子,基本偏向与于A/U结尾,但最优密码子存在差异。毛茛科和芍药科叶绿体基因组密码子偏好性的形成因素主要受自然选择的影响,且芍药科叶绿体基因组密码子偏好性受自然选择的影响大于毛茛科。基于叶绿体基因组全序列和基于叶绿体基因组CDS序列的系统进化关系表明,芍药科基于叶绿体基因组全序列和基于叶绿体基因组CDS序列的系统进化关系虽然部分不同,但都可以被划分为芍药组和牡丹组。毛茛科基于叶绿体基因组的系统进化关系不符合中国植物志分类关系,但支持把毛茛科划分为4亚科14族。系统进化分析结果也支持芍药科独立于毛茛科和毛茛目,被划分到虎耳草目,同时证明了叶绿体基因组作为超级DNA条形码的可行性。     

Evidence from simulation studies for selective constraints on the codon usage of the Angiosperm psbA gene

The codon usage of the Angiosperm psbA gene is atypical for flowering plant chloroplast genes but similar to the codon usage observed in highly expressed plastid genes from some other Plantae, particularly Chlorobionta, lineages. The pattern of codon bias in these genes is suggestive of selection for a set of translationally optimal codons but the degree of bias towards these optimal codons is much weaker in the flowering plant psbA gene than in high expression plastid genes from lineages such as certain green algal groups. Two scenarios have been proposed to explain these observations. One is that the flowering plant psbA gene is currently under weak selective constraints for translation efficiency, the other is that there are no current selective constraints and we are observing the remnants of an ancestral codon adaptation that is decaying under mutational pressure. We test these two models using simulations studies that incorporate the context-dependent mutational properties of plant chloroplast DNA. We first reconstruct ancestral sequences and then simulate their evolution in the absence of selection on codon usage by using mutation dynamics estimated from intergenic regions. The results show that psbA has a significantly higher level of codon adaptation than expected while other chloroplast genes are within the range predicted by the simulations. These results suggest that there have been selective constraints on the codon usage of the flowering plant psbA gene during Angiosperm evolution.